Octanoic acid kills Lucilia sericata (Diptera: Calliphoridae) by affecting two major defence systems: cuticular free fatty acids and immunocompetent cells.

This work examines the insecticidal activity of octanoic acid (C8:0), a short-chain fatty acid detected in entomopathogenic fungus - Conidiobolus coronatus medium, against Lucilia sericata larvae and adults. The LD50 value was calculated as 3.04±0.26 µg/mg (3040 mg/kg) of insect body mass, which places the compound in category 5 of acute toxicity (slightly hazardous). The presented research also describes its probable mechanism, with a particular focus on changes in two main insect defense mechanisms: (1) the composition of the cuticle (GC-MS analysis) and (2) immunocompetent cells (microscopic analysis of cultured hemocytes). More precisely, octanoic acid application resulted in changes in cuticular free fatty acid (FFA) profiles in both adults and larvae; generally, treatment increased short-chain FFAs, and a decrease of middle- and long-chain FFAs. Both in vivo and in vitro applications of octanoic acid resulted in vacuolisation, disintegration, and destruction of nets formed by plasmatocytes. As the compound has also previously been found to be toxic against Galleria mellonella, it appears to have lethal potential against insects in both the Orders Diptera and Lepidoptera, indicating it may have strong entomopathogenic potential. It is worth noting that octanoic acid is approved as a food additive with well-documented insecticidal activity, and hence may be a valuable component in the design of new insecticides that are safe for both humans and the environment.

The effects of APOE4 and familial Alzheimer's disease mutations on free fatty acid profiles in mouse brain are age- and sex-dependent.

APOE4 encoding apolipoprotein (Apo)E4 is the strongest genetic risk factor for Alzheimer's disease (AD). ApoE is key in intercellular lipid trafficking. Fatty acids are essential for brain integrity and cognitive performance and are implicated in neurodegeneration. We determined the sex- and age-dependent effect of AD and APOE4 on brain free fatty acid (FFA) profiles. FFA profiles were determined by LC-MS/MS in hippocampus, cortex, and cerebellum of female and male, young (≤3 months) and older (>5 months), transgenic APOE3 and APOE4 mice with and without five familial AD (FAD) mutations (16 groups; n = 7-10 each). In the different brain regions, females had higher levels than males of either saturated or polyunsaturated FFAs or both. In the hippocampus of young males, but not of older males, APOE4 and FAD each induced 1.3-fold higher levels of almost all FFAs. In young and older females, FAD and to a less extent APOE4-induced shifts among saturated, monounsaturated, and polyunsaturated FFAs without affecting total FFA levels. In cortex and cerebellum, APOE4 and FAD had only minor effects on individual FFAs. The effects of APOE4 and FAD on FFA levels and FFA profiles in the three brain regions were strongly dependent of sex and age, particularly in the hippocampus. Here, most FFAs that are affected by FAD are similarly affected by APOE4. Since APOE4 and FAD affected hippocampal FFA profiles already at young age, these APOE4-induced alterations may modulate the pathogenesis of AD.

Exploring the effect of part differences on metabolite molecule changes in refrigerated pork: Identifying key metabolite compounds and their conversion pathways.

Effect of part differences on metabolite molecule alterations in refrigerated pork was investigated. A metabolomics methodology combined with chemometric analysis was successfully established to identify key compounds and their conversion pathways, including precursors and volatile metabolites, in the Longissimus lumborum as well as the breast and flank stored for 11 days. In total, 12 discriminative precursors were identified using the Short Time-series Expression Miner. In tandem with Random Forest and ANOVA analyses, nine volatile metabolites were identified as key compounds that could be attributed to differences in pork sections. Bidirectional orthogonal partial least squares analysis revealed a potential correlation between these key metabolites and discriminative precursors. Metabolic pathway enrichment analysis demonstrated that the primary metabolic process affected by variations in pork sections is linoleic acid metabolism, which participates in the metabolism of cysteine and glutamic acid to produce methoxy-phenyl-oxime. This study provides valuable insights into the identification of differential metabolites.

Unravelling metabolic factors impacting iNKT cell biology in obesity.

Obesity and related diseases have reached epidemic proportions and continue to rise. Beyond creating an economical burden, obesity and its co-morbidities are associated with shortened human life expectancy. Despite major advances, the underlying mechanisms of obesity remain not fully elucidated. Recently, several studies have highlighted that various immune cells are metabolically reprogrammed in obesity, thereby profoundly affecting the immune system. This sheds light on a new field of interest: the impact of obesity-related systemic metabolic changes affecting immune system that could lead to immunosurveillance loss. Among immune cells altered by obesity, invariant Natural Killer T (iNKT) cells have recently garnered intense focus due to their ability to recognize lipid antigen. While iNKT cells are well-described to be affected by obesity, how and to what extent immunometabolic factors (e.g., lipids, glucose, cytokines, adipokines, insulin and free fatty acids) can drive iNKT cells alterations remains unclear, but represent an emerging field of research. Here, we review the current knowledge on iNKT cells in obesity and discuss the immunometabolic factors that could modulate their phenotype and activity.

Do concentrations of non-esterified fatty acids change during gestation and lactation in healthy bitches?

During the gestation and lactation period, the energy demand in pregnant and lactating bitches is elevated. Non-esterified fatty acids (NEFAs) are utilized either directly from the fed diet or from body fat storage. High NEFA concentration in the blood plasma leads to an increased risk for diseases. Therefore, measuring blood NEFA concentrations may be an indicator for a period of scarcity. The aim of this study is to explore if serum NEFA concentrations in healthy bitches change during gestation and lactation. Healthy pregnant and lactating bitches were sampled on three appointed dates around parturition. NEFA values were examined with a multiparameter clinical chemistry analyser. All statistical analyses were performed using R. Overall, 38 bitches were enrolled in the study. Twenty-one bitches were sampled on all three appointed dates. The median NEFA concentration antepartum was 0.73 mmol/L (IQR: 0.59, 1.01); during peak lactation, it was 0.57 mmol/L (IQR: 0.44, 0.82); and around weaning, it was 0.58 mmol/L (IQR: 0.46, 0.73). NEFA concentrations rose slightly with litter size in late gestation. Body condition score had no influence on observed NEFA values. We conclude that NEFA concentrations widely remain within reference ranges in well-fed pregnant and lactating bitches. Nevertheless, they may be a valuable parameter to assess the actual metabolic status of malnourished pregnant and lactating bitches.

The fate of intracellular S1P regulates lipid droplet turnover and lipotoxicity in pancreatic beta-cells.

Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-ßH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA.

Targeted metabolomic analysis of serum free fatty acids: Lipidomics disturbance in patients with superior limbic keratoconjunctivitis.

This study aimed to identify and quantify free fatty acids (FFAs), secretory phospholipase A2 group IIa (sPLA2-IIa) and cytosolic phospholipase A2 (cPLA2) in serum of superior limbic keratoconjunctivitis (SLK) patients and explored the association between FFAs, sPLA2-IIa and cPLA2 variations and SLK. Targeted metabolomic analysis of FFAs in serum was performed by gas chromatography tandem mass spectrometry (GC-MS/MS) analysis on 16 SLK patients (43.88 ± 7.88 years; female: 62.50%) and 25 healthy controls (43.12 ± 7.88 years; female: 64.00%). Qualitative and absolute quantitative results of FFAs were obtained and classified according to gender and thyroid tests. Differential lipid metabolites, metabolomic pathways and biomarkers were further evaluated. The serum sPLA2-IIa and cPLA2 were determined by enzyme linked immunosorbent assay (ELISA). Among 40 FFAs identified, 6 FFAs showed significant changes (P < 0.05) in SLK patients, including 4 decreased and 2 increased. They were mainly related to unsaturated fatty acid biosynthesis, α-linolenic acid and linoleic acid metabolism, and fatty acid biosynthesis. When dividing the data by gender or abnormal thyroid tests, some comparable FFAs alterations displayed in SLK patients. The ROC analysis revealed that the AUC values of linoleic acid, γ-linolenic acid, cis-8,11,14-eicosatrienoic acid, stearic acid, and palmitic acid, were all greater than 0.8. The serum concentrations of sPLA2-IIa and cPLA2 in patients with SLK were significantly higher than that in healthy controls. Lipidomics disturbance might be the potential mechanism of SLK. Serum FFA biomarkers associated with SLK have potential for the diagnosis and treatment of the disease.

Free Fatty Acids and Free Fatty Acid Receptors: Role in Regulating Arterial Function.

The metabolic network's primary sources of free fatty acids (FFAs) are long- and medium-chain fatty acids of triglyceride origin and short-chain fatty acids produced by intestinal microorganisms through dietary fibre fermentation. Recent studies have demonstrated that FFAs not only serve as an energy source for the body's metabolism but also participate in regulating arterial function. Excess FFAs have been shown to lead to endothelial dysfunction, vascular hypertrophy, and vessel wall stiffness, which are important triggers of arterial hypertension and atherosclerosis. Nevertheless, free fatty acid receptors (FFARs) are involved in the regulation of arterial functions, including the proliferation, differentiation, migration, apoptosis, inflammation, and angiogenesis of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). They actively regulate hypertension, endothelial dysfunction, and atherosclerosis. The objective of this review is to examine the roles and heterogeneity of FFAs and FFARs in the regulation of arterial function, with a view to identifying the points of intersection between their actions and providing new insights into the prevention and treatment of diseases associated with arterial dysfunction, as well as the development of targeted drugs.

Excessive free fatty acid sensing in pituitary lactotrophs elicits steatotic liver disease by decreasing prolactin levels.

The pituitary is the central endocrine gland with effects on metabolic dysfunction-associated steatotic liver disease (MASLD). However, it is not clear whether the pituitary responds to free fatty acid (FFA) toxicity, thus dysregulating hepatic lipid metabolism. Here, we demonstrate that decreased prolactin (PRL) levels are involved in the association between FFA and MASLD based on a liver biospecimen-based cohort. Moreover, overloaded FFAs decrease serum PRL levels, thus promoting liver steatosis in mice with both dynamic diet intervention and stereotactic pituitary FFA injection. Mechanistic studies show that excessive FFA sensing in pituitary lactotrophs inhibits the synthesis and secretion of PRL in a cell-autonomous manner. Notably, inhibiting excessive lipid uptake using pituitary stereotaxic virus injection or a specific drug delivery system effectively ameliorates hepatic lipid accumulation by improving PRL levels. Targeted inhibition of pituitary FFA sensing may be a potential therapeutic target for liver steatosis.

Innovative Alkanediol-Based Eutectic Solvents for Extracting/Pre-Formulating Dermatologically Valuable Free Fatty Acids from Spirulina and Porphyridium Cakes.

The growing demand for phycobiliproteins from microalgae generates a significant volume of by-products, such as extraction cakes. These cakes are enriched with products of interest for the cosmetics market, namely free fatty acids, particularly polyunsaturated (PUFA). In this work, two cakes, one of spirulina and one of Porphyridium cruentum, were valorized using innovative natural hydrophobic deep eutectic solvents (NaDES) based on alkanediols. The most promising NaDES, as determined by physicochemical properties and screening, are mixtures of alkanediols and fatty acids. These include the mixtures of 1,3-propanediol and octanoic acid (1:5, mol/mol) and 1,3-propanediol and octanoic and decanoic acid (1:3:1, mol/mol). Two extractive processes were implemented: ultrasound-assisted extraction and an innovative mechanical process involving dual asymmetric centrifugation. The second process resulted in the production of extracts significantly enriched in PUFA, ranging from 65 to 220 mg/g dry matter with the two cakes. The extracts and NaDES demonstrated good safety with respect to epidermal keratinocyte viability (>80% at 200 µg/mL). The study of their impact on commensal and pathogenic cutaneous bacteria demonstrated significant effects on the viability of Staphylococcus aureus and Staphylococcus epidermidis (>50% decrease at 200 µg/mL) while preserving Corynebacterium xerosis and Cutibacterium acnes. These results highlight the potential of valorizing these co-products using alkanediol-based NaDES, in a strategy combining an active vector (NaDES) and a growth regulator extract, for the management of cutaneous dysbiosis involving staphylococci.

In situ interaction of pea peptides and bile salts under in vitro digestion: Potential impact on lipolysis.

The present work evaluated how a native pea protein isolate (PPI) affects the key roles carried out by bile salts (BS) in lipid digestion by means of the in vitro static INFOGEST protocol. Two gastric residence times were evaluated (10 and 60 min), and then the peptides obtained (GPPP) were mixed with BS at physiological concentration in simulated intestinal fluid to understand how they interact with BS both at the bulk and at the interface. Both GPPP give rise to a film with a predominant viscous character that does not constitute a barrier to the penetration of BS, but interact with BS in the bulk duodenal fluid. When the peptides flushing from the stomach after the different gastric residence times undergo duodenal digestion, it was found that for the longer gastric residence time the percentage of soluble fraction in the duodenal phase, that perform synergistically with BS micelles, was twice that of the lower residence time, leading to an increase in the solubilization of oleic acid. These results finally lead to a greater extent of lipolysis of olive oil emulsions. This work demonstrates the usefulness of in vitro models as a starting point to study the influence of gastric residence time of pea protein on its interaction with BS, affecting lipolysis. Pea proteins were shown to be effective emulsifiers that synergistically perform with BS improving the release and bioaccessibility of bioactive lipids as olive oil.

A new chemical derivatization reagent sulfonyl piperazinyl for the quantification of fatty acids using LC-MS/MS.

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80 % (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

Similar insulin regulation of splanchnic FFA and VLDL-TG in men with nonalcoholic hepatic steatosis and steatohepatitis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) versus 38 (18-58) µmol/min; simple steatosis: 62 (46-78) versus 45 (25-65) µmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P <0.05 time-effect) in both groups. Insulin-mediated glucose disposal was similar in the two groups (P = 0.54). Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from visceral adipose tissue lipolysis delivered to the liver. These results demonstrate that the splanchnic balances of FFA and VLDL-TG do not differ between obese men with NASH and those with simple steatosis.

Enhancing rheology and reducing lipid digestion of oil-in-water emulsions using controlled aggregation and heteroaggregation of soybean protein isolate-peach gum microspheres.

There is a growing interest in developing highly viscous lipid foods using plant protein and polysaccharide gum-based emulsion technology. However, gaps remain in understanding the rheological, microstructural, and digestive properties of plant proteins like soybean protein isolate (SPI) in combination with various gums. This study investigates how combining SPI and peach gum (PG) affects rheology and lipolysis of oil-in-water (O/W) emulsions containing 20 wt% soybean oil. Emulsions with varying SPI and PG compositions including SPI-PG single and SPI/PG mixed droplet systems were prepared. Heating induced alterations in viscosity (e.g., SPI-PG from 14.88 to 90.27 Pa·s and SPI/PG from 9.66 to 85.32 Pa·s) and microstructure revealing aggregate formation at oil-water interface. The viscosity decreased significantly from the oral to intestinal phase (SPI-PG: 28.10 to 0.19 Pa·s, SPI/PG: 21.27 to 0.10 Pa·s). These changes affected lipid digestion, notably in SPI-PG and SPI/PG emulsions where a compact interface hindered lipolysis during digestion. Interestingly, free fatty acid (FFA) release during small intestinal phase followed a different order: SPI (82.51 %) > SPI-PG (70.77 %) > SPI/PG (63.60 %) > PG (56.09 %). This study provides insights into creating highly viscous O/W spreads with improved rheology, stability, and delayed lipid digestion, offering potential benefits in food product formulation.

Intestinal GSTpi deficiency exacerbates the severity of experimental hyperlipidemic acute pancreatitis.

Intestinal dysfunction plays a pivotal role in the development of acute pancreatitis (AP), however, the underlying mechanisms of intestinal dysfunction on severity of hyperlipidemic acute pancreatitis (HLAP) are still unclear. Herein, we explored the role of intestinal function on the severity of HLAP. We found that HLAP patients exhibit higher lipid and inflammatory response than AP patients. Hyperlipidemia significantly elevates serum lipids and worsen pancreatic damage in AP mice. In addition, significant exacerbated intestinal barrier damage and inflammation were observed in experimental HLAP mice, as evidenced by increased serum amylase and lipase levels, and pancreatic edema. Further, RNA-Seq showed that a markedly decrease of glutathione S-transferase pi (GSTpi) in colonic tissue of HLAP mice compared with AP mice, accompanied with increased serum lipopolysaccharides level. However, colonic GSTpi overexpression by adeno-associated virus significantly attenuated intestinal damage and subsequent pancreatic inflammation in HLAP mice. Mechanistically, GSTpi mitigated HLAP-mediated colonic NLRP3 inflammasome activation and barrier dysfunction. These results suggest that intestinal GSTpi deficiency exacerbates the severity of experimental HLAP, providing new insights for the clinical treatment of HLAP.

The impacts of cold plasma on the taste and odor formation of dried silver carp products.

This study investigated non-thermal pretreatment (cold plasma, CP) on the flavor (taste and odor) profiles of dried fish products. CP treatment of 5 min contributed to accumulation of umami nucleotides adenosine 5'-monophosphate (AMP) from 30.96 to 40.82 µg/g and inosine 5'-monophosphate (IMP) from 2009.29 to 2132.23 µg/g, and significant reduction of bitter hypoxanthine ribonucleoside (HxR) and hypoxanthine (Hx), respectively (P < 0.05) in the dried fish products. A noticeable enhancement in sweet glycine (from 429.41 to 490.03 mg/100 g) and umami glutamic acid (from 55.68 to 67.76 mg/100 g) accompanied with the CP treatment (P < 0.05) based on taste activity value (TAV > 1). And the characteristic odor volatiles (nonanal, hexanal and 1-octen-3-ol) were strengthened 2.13-, 2.16- and 2.17- folds, respectively (P < 0.05). The results of equivalent umami concentration and Gibbs free energy calculation, combining with the correlation analysis, indicate that nucleotides and free amino acids synergically enhanced the taste improvement of dried fish products. Moderate lipids oxidation favored the formation of characteristic volatiles. The CP pretreatment offered new strategies for enhancing flavor of dried fish products.

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy.

A stable isotope dilution-liquid chromatography-tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water-acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL-1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL-1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0-14.0 ng mL-1; the limits of quantification (LOQs) were in the range of 8.0-45.0 ng mL-1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7-9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+ binding.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Association of circulating long-chain free fatty acids and incident diabetes risk among normoglycemic Chinese adults: a prospective nested case-control study.

BACKGROUND: Long-chain free fatty acids (FFAs) are associated with risk of incident diabetes. However, a comprehensive assessment of the associations in normoglycemic populations is lacking. OBJECTIVES: Our study aimed to comprehensively investigate the prospective associations and patterns of FFA profiles with diabetes risk among normoglycemic Chinese adults. METHODS: This is a prospective nested case-control study from the China Cardiometabolic Disease and Cancer Cohort (4C) study. We quantitatively measured 53 serum FFAs using a targeted metabolomics approach in 1707 incident diabetes subjects and 1707 propensity score-matched normoglycemic controls. Conditional logistic regression models were employed to estimate odds ratios (ORs) for associations. Least Absolute Shrinkage and Selection Operator (LASSO) penalty regression and quantile g-computation (qg-comp) analyses were implemented to estimate the association between multi-FFA exposures and incident diabetes. RESULTS: The majority of odd-chain FFAs exhibited an inverse association with incident diabetes, wherein the ORs per SD increment of all 7 saturated fatty acids (SFAs), monounsaturated fatty acid (MUFA) 15:1, and polyunsaturated fatty acid (PUFA) 25:2 were ranging from 0.79 to 0.88 (95% CIs ranging between 0.71 and 0.97). Even-chain FFAs comprised 99.3% of total FFAs and displayed heterogeneity with incident diabetes. SFAs with 18-26 carbon atoms are inversely linked to incident diabetes, with ORs ranging from 0.81 to 0.86 (95% CIs ranging between 0.73 and 0.94). MUFAs 26:1 (OR: 0.85; 95% CI: 0.76, 0.94), PUFAs 20:4 (OR: 0.84; 95% CI: 0.75, 0.94), and 24:2 (OR: 0.87; 95% CI: 0.78, 0.97) demonstrated significant associations. In multi-FFA exposure model, 24 FFAs were significantly associated with incident diabetes, most of which were consistent with univariate results. The mixture OR was 0.78 (95% CI: 0.61, 0.99; P = 0.04159). Differential correlation network analysis revealed pre-existing perturbations in intraclass and interclass FFA coregulation before diabetes onset. CONCLUSIONS: These findings underscore the variations in diabetes risk associated with FFAs across chain length and unsaturation degree, highlighting the importance of recognizing FFA subtypes in the pathogenesis of diabetes.

Expression of free fatty acid receptor 2 in normal and neoplastic tissues.

OBJECTIVE: Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study was to comprehensively characterise the expression profile of FFAR2 in a large series of human normal and neoplastic tissues using immunohistochemistry thus providing a basis for further in-depth investigations into its potential diagnostic or therapeutic importance. METHODS: We developed a novel rabbit polyclonal anti-FFAR2 antibody, 0524, directed against the C-terminal region of human FFAR2. Antibody specificity was confirmed via Western blot analyses and immunocytochemistry using the FFAR2-expressing cell line BON-1 and FFAR2-specific small interfering RNA as well as native and FFAR2-transfected HEK-293 cells. The antibody was then used for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic human tissues. RESULTS: In normal tissues, FFAR2 was mainly present in distinct cell populations of the cerebral cortex, follicular cells and C cells of the thyroid, cardiomyocytes of the heart, bronchial epithelia and glands, hepatocytes and bile duct epithelia of the liver, gall bladder epithelium, exocrine and ß-cells of the endocrine pancreas, glomerular mesangial cells and podocytes as well as collecting ducts of the kidney, intestinal mucosa (particularly enteroendocrine cells), prostate epithelium, seminiferous tubules of the testicles, and placental syncytiotrophoblasts. In neoplastic tissues, FFAR2 was particularly prevalent in papillary thyroid carcinomas, parathyroid adenomas, and gastric, colon, pancreatic, hepatocellular, cholangiocellular, urinary bladder, breast, cervical, and ovarian carcinomas. CONCLUSIONS: We generated and characterised a novel rabbit polyclonal anti-human FFAR2 antibody that is well-suited for visualising FFAR2 expression in human routine pathology tissues. This antibody is also suitable for Western blot and immunocytochemistry experiments. To our knowledge, this antibody enabled the first broad FFAR2 protein expression profile in various normal and neoplastic human tissues.